Method for the identification of a risk for a thrombogenic disorder by determining the TAFI-Ile347 polymorphism

ABSTRACT

The present invention is directed to a method identifying a risk for a thrombogenic disorder including, without limitation, atrial fibrillation, stroke, prolonged intermitted neurological deficit (PRIND), transitory ischemic attack (TIA), atherosclerotic cerebrovascular disease (CVD) and/or coronary heart disease, as well as to a method for selecting patients with a risk for a thrombogenic disorder, to a method for identifying a pharmaceutical for the therapy or prophylaxis of a thrombogenic disorder as well as to a method for producing a medicament and a diagnostic by employing the TAFI-Ile347 polymorphism.

The present invention is directed to a method identifying a risk for athrombogenic disorder including, without limitation, atrialfibrillation, stroke, prolonged intermitted neurological deficit(PRIND), transitory ischemic attack (TIA), atheroscleroticcerebrovascular disease (CVD) and/or coronary heart disease, as well asto a method for selecting patients with a risk for a thrombogenicdisorder, to a method for identifying a pharmaceutical for the therapyor prophylaxis of a thrombogenic disorder as well as to a method forproducing a medicament and a diagnostic by employing the TAFI-Ile347polymorphism.

The Thrombin-Activable Fibirinolysis Inhibitor (TAFI) is a known plasmazymogen synthesized in the liver as a prepropeptide consisting of 423amino acids with a molecular weight of 55 kDa (FIG. 1). Theprepropeptide contains a 22 amino acids long signal peptide (amino acidsNo. 1-22), a 92 amino acids long activation peptide and a 309 aminoacids long catalytic domain. The nucleic acid sequence contains 1723nucleotides (FIG. 2). The protein sequence accession number (NCBIprotein database) of TAFI is NP_(—)001863, the nucleotide sequenceaccession number (NCBI nucleotide database) is NM_(—)001872 and theaccession number for TAFI information in OMIM (Online MendelianInheritance in Man™) is 603101.

TAFI is activated by thrombin, plasmin or the thrombin/thrombomodulincomplex. After processing, TAFI attenuates clot lysis by removing lysineresidues from a fibrin clot. Activated and processed TAFI is unstable at37° C. and has a half-life of about 8 minutes. Therefore, TAFI plays acentral role in homeostasis where it functions as a potent fibrinolysisinhibitor. The human TAFI gene has been mapped to chromosome 13q14.11.It consists of 11 exons and spans a genomic region of about 48 kb inlength (Boffa et al. (1999) Biochemistry, 38, 6547-6558).

Genetic analyses of the TAFI gene in humans revealed several variablenucleotides (SNPs, Single Nucleotide Polymorphisms) in the promoter andthe coding region. For SNPs in the promoter region of the TAFI gene ithas been shown, that some of these polymorphisms are associated withaltered TAFI protein levels in the blood (Franco et al. (2001)Haematologica, 86, 510-517; Henry et al. (2001) Blood, vol. 97, no. 7,2053-2058).

Recently two SNPs have been identified in the coding region of the TAFIgene leading to an amino acid exchange in the corresponding TAFIprotein, these polymorphism are T169A (T=Threonine (Thr) at position 169to A=Alanine (Ala)) and T347I (Threonine at position 347 to I=Isoleucine(Ile)). The TAFI-Ile347 variant seems to display an extended half-lifefrom 8 minutes to 15 minutes and seems to exhibit an enhancedantifibrinolytic potential of 60% (Brouwers et al. (2001) Blood, vol.98, no. 6, 1992-1993; Schneider et al. (2001) J. Biological Chemistry,vol. 277, no. 2, 1021-1030) under the tested in vitro conditions.Variations at position 169 of the TAFI protein do not seem to have anyeffect on the antifibrinolytic potential of TAFI (Schneider et al.(2001) supra). However, currently no data are available about theclinical effects, if any, of the described polymorphisms including theTAFI-Ile347 variant for thrombogenic or other disorders.

According to the present invention it has been found that in particularindividuals with a TAFI-Ile347 polymorphism have an increased risk forstroke and transitory ischemic attack (TIA). Therefore, the geneticTAFI-Ile347 polymorphism can be used as a genetic marker, e.g. for theidentification of a risk for a thrombogenic disorder, for the selectionof patients with a risk for a thrombogenic disorder, e.g. in clinicalstudies, for the identification of a pharmaceutical for the therapyand/or prophylaxis of a thrombogenic disorder, for the production of amedicament for the preventive and/or therapeutic treatment of athrombogenic disorder, for the production of a diagnostic for theidentification of a thrombogenic disorder and/or for the adaptation ofthe dosage of a medicament for the treatment and/or prophylaxis of athrombogenic disorder.

Therefore, one embodiment of the present invention is directed to an invitro or in vivo method for identifying a risk for a thrombogenicdisorder including, without limitation, the risk for an atrialfibrillation, stroke, prolonged intermitted neurological deficit(PRIND), transitory ischemic attack (TIA) and, atheroscleroticcerebrovascular disease (CVD) and/or coronary heart disease, wherein themethod comprises determining in a sample the presence of an amino acidexchange from threonine to isoleucine at position 347 of theThrombin-Activable Fibrinolysis Inhibitor (TAFI) with the amino acidsequence according to SEQ ID NO: 1 (TAFI-Ile347). Because diabeticsoften show hypofibrinolysis the sample is preferably from a diabetic.Generally, the sample is a cell or a body fluid, e.g. blood or an animalor a human cell, isolated from an animal or human body.

TIA, PRIND and stroke are neurological deficits with a sudden onset dueto a vascular disease such as a thrombogenic disorder which differs onlyin the time period of reconvalescence and in the severity. For example,for TIA the neurologic deficits generally last less than 24 hours and nopermanent brain damage results. For PRIND the neurologic deficitsgenerally last for more than 24 hours without permanent brain damages. Astroke generally results in a permanent brain damage.

The TAFI-Ile347 polymorphism can be generally determined by methodsknown to a person skilled in the art. One method is to determine theamino acid exchange by amino acid sequencing, e.g. standard proteindegradation or analysis of protein sequence fragments with massspectrometry, enzymatic treatment of the protein and subsequent analysisof degradation products or by means of a binding protein or a bindingpeptide or aptamer, specifically directed against TAFI-Ile347, inparticular by means of an antibody, an antigen-binding part of anantibody and/or a protein-scaffold, preferably an anticalin.

According to the present invention the term “binding protein” or“binding peptide” refers to a class of proteins or peptides whichspecifically bind TAFI or TAFI-Ile347 including, without limitation,monospecific polyclonal or monoclonal antibodies, antibody fragments andprotein scaffolds specifically directed against TAFI or TAFI-Ile347,e.g. anticalins which are specifically directed against TAFI-Ile347. Theterm “specifically” means that the binding protein or binding peptidediscriminates between TAFI-Ile347 and other polymorphisms at amino acidposition No. 347 of TAFI, in particular TAFI-Thr347.

The determination of other polymorphisms at amino acid position No. 347of TAFI, in particular TAFI-Thr347 may be used as a reference or controlin the method of the present invention.

The procedure for preparing an antibody or antibody fragment is effectedin accordance with methods which are well known to the skilled person,e.g. by immunizing a mammal, for example a rabbit, with TAFI orTAFI-Ile347, where appropriate in the presence of, for example, Freund'sadjuvant and/or aluminium hydroxide gels (see, for example, Diamond, B.A. et al. (1981) The New England Journal of Medicine: 1344-1349). Thepolyclonal antibodies which are formed in the animal as a result of animmunological reaction can subsequently be isolated from the blood usingwell known methods and, for example, purified by means of columnchromatography. Monoclonal antibodies can, for example, be prepared inaccordance with the known method of Winter & Milstein (Winter, G. &Milstein, C. (1991) Nature, 349, 293-299).

According to the present invention the term “antibody” or “antibodyfragment” is also understood as meaning antibodies or antigen-bindingparts thereof, which have been prepared recombinantly and, whereappropriate, modified, such as chimeric antibodies, humanizedantibodies, multifunctional antibodies, bispecific or oligospecificantibodies, single-stranded antibodies and F(ab) or F(ab)₂ fragments(see, for example, EP-B1-0 368 684, U.S. Pat. No. 4,816,567, U.S. Pat.No. 4,816,397, WO 88/01649, WO 93/06213 or WO 98/24884).

As an alternative to the classical antibodies it is also possible, forexample, to use protein scaffolds against TAFI or TAFI-Ile347, e.g.anticalins which are based on lipocalin (Beste et al. (1999) Proc. Natl.Acad. Sci. USA, 96, 1898-1903). The natural ligand-binding sites of thelipocalins, for example the retinol-binding protein or the bilin-bindingprotein, can be altered, for example by means of a “combinatorialprotein design” approach, in such a way that they bind to selectedhaptens, here to TAFI or TAFI-Ile347 (Skerra, 2000, Biochim. Biophys.Acta, 1482, 337-50). Other known protein scaffolds are known as beingalternatives to antibodies for molecular recognition (Skerra (2000) J.Mol. Recognit., 13, 167-187).

Aptamers are nucleic acids which bind with high affinity to apolypeptide, here TAFI or TAFI-ILe347. Aptamers can be isolated byselection methods such as SELEX (see e.g. Jayasena (1999) Clin. Chem.,45, 1628-50; Klug and Famulok (1994) M. Mol. Biol. Rep., 20, 97-107;U.S. Pat. No. 5,582,981) from a large pool of different single-strandedRNA molecules. Aptamers can also be synthesized and selected in theirmirror-image form, for example as the L-ribonucleotide (Nolte et al.(1996) Nat. Biotechnol., 14, 1116-9; Klussmann et al. (1996) Nat.Biotechnol., 14, 1112-5). Forms which have been isolated in this wayenjoy the advantage that they are not degraded by naturally occurringribonucleases and, therefore, possess greater stability.

Another method to determine the TAFI-Ile347 polymorphism is the analysisof the TAFI gene, in particular the nucleic acid sequence at position1064 for the detection of the nucleotide exchange from cytidine tothymidine. In general, the determination of the nucleotide exchange canbe carried out by nucleic acid sequencing, e.g. pyrosequencing,sequencing methods using radio-labelled nucleotides or nucleotideslabelled with a fluorescent dye, primer extension assay, analysis ofsequence fragments with mass spectrometry, or by means of an antibody,an antigen-binding part of an antibody or a protein-scaffold, preferablyan anticalin and/or a complementary nucleic acid, specifically directedagainst the mutation in the TAFI gene.

In this respect the term “specifically” means that the antibody, anantigen-binding part of an antibody or a protein-scaffold, preferably ananticalin, and/or a complementary nucleic acid discriminates between theTAFI-Ile347 gene and other polymorphisms of the nucleotide codon foramino acid position No. 347 of TAFI, in particular TAFI-Thr347. Thepreferred nucleotide exchange for the polymorphism of the amino acid atposition No. 347 is the nucleotide at position 1064.

The complementary nucleic acids, which are preferably single strandedDNA molecules, can be synthesized chemically, e.g. in accordance withthe phosphotriester method (see, for example, Uhlmann, E. & Peyman, A.(1990) Chemical Reviews, 90, 543-584). These complementary nucleic acidscan be used as hybridization probes, e.g. on DNA microarrays, or asamplification probes, e.g. in the TaqMan® analysis (Taqman® Laboratory)which is a fluorogenic 5′ nuclease assay.

The antibody, antigen-binding part of an antibody or theprotein-scaffold can be produced accordingly as described above.

In any case it is advantageous for the method of the present inventionto additionally determine other potential polymorphisms at amino acidposition 347 of TAFI, e.g. TAFI-Thr347, as a reference or controlpreferably with a method as described above.

Generally, with the method of the present invention an increased riskfor a vascular disorder including, without limitation, atrialfibrillation, stroke, prolonged intermitted neurological deficit(PRIND), transitory ischemic attack (TIA), atheroscleroticcerebrovascular disease (CVD) and/or coronary heart disease compared toa reference or control can be identified which may lead to aprophylactic and/or therapeutic treatment of an individual, e.g. adiabetic, or to the adaptation of the dosage of a pharmaceutical to beadministered as described further below.

Therefore, another embodiment of the present invention is an in vitro orin vivo method for selecting patients with a risk for a thrombogenicdisorder including, without limitation, the risk for an atrialfibrillation, stroke, prolonged intermitted neurological deficit(PRIND), transitory ischemic attack (TIA), atheroscleroticcerebrovascular disease (CVD) and/or coronary heart disease, wherein themethod comprises determining in a sample, preferably from a diabetic,the presence of an amino acid exchange from threonine to isoleucine atposition 347 of the Thrombin-Activable Fibrinolysis Inhibitor (TAFI)with the amino acid sequence according to SEQ ID NO: 1.

The present method for selecting patients with a risk for a vasculardisorder is characterized and can be carried out in the same way as themethod for identifying a risk for a vascular disorder described above.

Another embodiment of the present invention is directed to a method foridentifying a pharmaceutical, preferably an inhibitor of TAFI-Ile347,for the therapy and/or prophylaxis of a thrombogenic disorder including,without limitation, an atrial fibrillation, stroke, prolongedintermitted neurological deficit (PRIND), transitory ischemic attack(TIA) and, atherosclerotic cerebrovascular disease (CVD) and/or coronaryheart disease, wherein the method comprises the steps of:

-   (a) providing TAFI-Ile347 or the TAFI-Ile347 gene,-   (b) providing a test compound, and-   (c) measuring or detecting the influence of the test compound on    TAFI-Ile347 or the TAFI-Ile347 gene.

According to the present invention the term “inhibitor” refers to abiochemical or chemical compound which inhibits or reduces the zymogenicactivity of TAFI-Ile347, in particular the removal of lysine residuesfrom a fibrin clot, and/or reduces the half-life of TAFI-Ile347,especially by at least about 20% to about 50%, preferably at least byabout 30% to about 45%, in particular by about 45%. The term “about”means generally an error range of +/−20%, especially +/−10%, preferably+/−5%.

In general, TAFI-Ile347 or the TAFI-Ile347 gene is provided e.g. in anassay system and brought directly or indirectly into contact with a testcompound, in particular a biochemical or chemical test compound, e.g. inthe form of a chemical compound library. Then, the influence of the testcompound on TAFI-Ile347 or the TAFI-Ile347 gene is measured or detected.Thereafter, suitable inhibitors can be analyzed and/or isolated. For thescreening of chemical compound libraries, the use of high-throughputassays are preferred which are known to the skilled person or which arecommercially available.

According to the present invention the term “chemical compound library”refers to a plurality of chemical compounds that have been assembledfrom any of multiple sources, including chemically synthesized moleculesand natural products, or that have been generated by combinatorialchemistry techniques.

In general, the influence of the test compound on TAFI-Ile347 or theTAFI-Ile347 gene is measured or detected in a heterogeneous orhomogeneous assay. As used herein, a heterogeneous assay is an assaywhich includes one or more washing steps, whereas in a homogeneous assaysuch washing steps are not necessary. The reagents and compounds areonly mixed and measured.

Suitable functional assays may be based on the gene expression ofTAFI-Ile347. In the presence of a biochemical or chemical compound to betested as an inhibitor of TAFI-Ile347 gene expression, the directinhibition can be measures by means generally known to a skilled personand as described above. For example, in order to measure the enzymaticactivity of TAFI (e.g. as a reference or control) and/or TAFI-Ile347 theclot lysis and/or the removal of lysin residues from fibrin or generallyits carboxypeptidase activity using e.g. the synthetic carboxypeptidasesubstrate anisylazoformyllysine can be measured in an appropriate assayknown to a person skilled in the art (see e.g. Schneider, M. et al.(2002) supra).

Heterogeneous assays are, for example, ELISA (enzyme linked immunosorbent assay), DELFIA, SPA and flashplate assays.

ELISA is a generally known assay which uses an enzyme as the markermolecule, e.g. a peroxidase colour reaction is initiated by addition ofthe peroxidase substrate and the optical density is measured in asuitable densitometer.

DELFIA (dissociation enhanced lanthanide fluoro immuno assay)-basedassays are solid phase assay. The antibody is usually labelled withEuropium or another lanthanide and the Europium fluorescence is detectedafter having washed away un-bound Europium-labelled antibodies.

SPA (scintillation proximity assay) and the flashplate assay usuallyexploit biotin/avidin interactions for capturing radiolabeledsubstrates. Generally the reaction mixture includes a biotinylatedpeptide substrate. After the reaction, the biotinylated peptides arecaptured by streptavidin. In the SPA detection, streptavidin is bound onscintillant containing beads whereas in the flashplate detection,streptavidin is bound to the interior of the well of scintillantcontaining microplates. Once immobilized, the radiolabelled substrate isclose enough to the scintillant to stimulate the emission of light.

Alternative homogeneous assays are, for example, TR-FRET, FP, ALPHA, EFCand gene assays.

TR-FRET (time-resolved fluorescence resonance energy transfer)-basedassays are assays which usually exploit the fluorescence resonanceenergy transfer between Europium and APC, a modified allophycocyanin, orother dyes with overlapping spectra such as Cy3/Cy5 or Cy5/Cy7 (Schobel,U. et al. (1999) Bioconjugate Chem. 10, 1107-1114). After excitatione.g. of Europium with light at 337 nm, the molecule fluoresces at 620nm. But if this fluorophore is close enough to APC, the Europium willtransfer its excitation energy to APC, which fluoresces at 665 nm. Afterthe reaction, Europium-labelled-antibodies are added along withstreptavidin-APC. The close proximity of the APC to the Europiumfluorophore will cause a quenching of the Europium fluorescence atbenefit of the APC fluorescence (FRET).

Fluorescence polarisation (FP)-based assays are assays which usepolarized light to excite fluorescent substrate peptides in solution.These fluorescent peptides are free in solution and tumble, causing theemitted light to become depolarised. When the substrate peptide binds toa larger molecule, however, its tumbling rates are greatly decreased,and the emitted light remains highly polarized.

ALPHA (amplified luminescent proximity homogenous)-based assays, areassays which rely on the transfer of singlet oxygen between donor andacceptor beads brought into proximity. Upon excitation at 680 nm,photosensitisers in donor beads convert ambient oxygen to singlet-stateoxygen, which diffuses up to a distance of 200 nm. Chemiluminescentgroups in the acceptor beads transfer energy to fluorescent acceptorswithin the bead, which then emits light at approximately 600 nm.

EFC (enzyme fragment complementation)-based assays or equivalent assayscan be used in particular for high-throughput screening of compounds.The EFC assay is based on an engineered β-galactosidase enzyme thatconsists of two fragments—the enzyme acceptor (EA) and the enzyme donor(ED). When the fragments are separated, there is no β-galactosidaseactivity, but when the fragments are together they associate(complement) to form active enzyme. The EFC assay utilizes an ED-analyteconjugate in which the analyte may be recognized by a specific bindingprotein, such as an antibody or receptor. In the absence of the specificbinding protein, the ED-analyte conjugate is capable of complementing EAto form active β-galactosidase, producing a positive luminescent signal.If the ED-analyte conjugate is bound by a specific binding protein,complementation with EA is prevented, and there is no signal. If freeanalyte is provided (in a sample), it will compete with the ED-analyteconjugate for binding to the specific binding protein. Free analyte willrelease ED-analyte conjugate for complementation with EA, producing asignal dependent upon the amount of free analyte present in the sample.

An example of a gene assay is the two-hybrid system assay (Fields andSternglanz (1994) Trends in Genetics, 10, 286-292; Colas and Brent(1998) TIBTECH, 16, 355-363). In this test, cells are transformed withexpression vectors which are expressing fusion proteins consisting ofthe polypeptide according to the invention and a DNA-binding domain of atranscription factor such as Gal4 or LexA. The transformed cellsadditionally contain a reporter gene whose promoter contains bindingsites for the corresponding DNA-binding domain. By transforming withanother expression vector which is expressing a second fusion proteinconsisting of a known or unknown polypeptide and an activation domain,for example from Gal4 or herpes simplex virus VP16, the expression ofthe reporter gene can be greatly increased if the second fusion proteininteracts with the polypeptide. Consequently this test system can beused for screening for biochemical or chemical compounds which inhibitan interaction between TAFI-Ile347 and its substrate, e.g. fibrin. Inthis way, it is possible to identify novel active compounds rapidly.

Another assay is based on solid phase-bound polypeptides such asTAFI-Ile347. Thus, a test compound, for example, contains a detectablemarker, for example, the compound can be radioactively labelled,fluorescence-labelled or luminescence-labelled. Furthermore, compoundscan be coupled to proteins which permit indirect detection, for exampleby means of enzymatic catalysis employing a peroxidase assay which usesa chromogenic substrate or by means of binding a detectable antibody.Another possibility is that of investigating the solid phase-boundprotein complexes by means of mass spectrometry (SELDI). Changes in theconformation of e.g. TAFI-Ile347 during its activation as the result ofinteraction with a test substance can be detected, for example, by thechange in the fluorescence of an endogenous tryptophan residue in thepolypeptide.

The solid phase-bound polypeptides can also be part of an array. Methodsfor preparing such arrays using solid phase chemistry and photolabileprotecting groups are disclosed, for example, in U.S. Pat. No.5,744,305. These arrays can also be brought into contact with testcompound or compound libraries and tested for interaction, for examplebinding or changing conformation.

Advantageously the method of the present invention is carried out in arobotics system e.g. including robotic plating and a robotic liquidtransfer system, e.g. using microfluidics, i.e. channeled structured.

In another embodiment of the present invention, the method is carriedout in form of a high-through put screening system. In such a systemadvantageously the screening method is automated and miniaturized, inparticular it uses miniaturized wells and microfluidics controlled by aroboter.

Another embodiment of the present invention is directed to a method forproducing a medicament for the preventive and/or therapeutic treatmentof a thrombogenic disorder including, without limitation, an atrialfibrillation, stroke, prolonged intermitted neurological deficit(PRIND), transitory ischemic attack (TIA) and, atheroscleroticcerebrovascular disease (CVD) and/or coronary heart disease, wherein themethod comprises the steps of:

-   (a) identifying a pharmaceutical for the therapy and/or prophylaxis    of a thrombogenic disorder according to the method described above,-   (b) providing an adequate amount of the pharmaceutical identified    according to step (a), and-   (c) formulating the pharmaceutical with one or more pharmaceutically    acceptable carriers or auxiliary substances.

For the production of the medicament of the present invention theidentified pharmaceutical is usually formulated with one or morepharmaceutically acceptable carriers or auxiliary substances, such asphysiological buffer solution, e.g. sodium chloride solution,demineralized water, stabilizers, such as protease or nucleaseinhibitors, preferably aprotinin, ε-aminocaproic acid or pepstatin A orsequestering agents such as EDTA, gel formulations, such as whitevaseline, low-viscosity paraffin and/or yellow wax, etc. depending onthe kind of administration.

Suitable further additives are, for example, detergents, such as, forexample, Triton X-100 or sodium deoxycholate, but also polyols, such as,for example, polyethylene glycol or glycerol, sugars, such as, forexample, sucrose or glucose, zwitterionic compounds, such as, forexample, amino acids such as glycine or in particular taurine or betaineand/or a protein, such as, for example, bovine or human serum albumin.Detergents, polyols and/or zwitterionic compounds are preferred.

The physiological buffer solution preferably has a pH of approx.6.0-8.0, especially a pH of approx. 6.8-7.8, in particular a pH ofapprox. 7.4, and/or an osmolarity of approx. 200-400 milliosmol/liter,preferably of approx. 290-310 milliosmol/liter. The pH of the medicamentis in general adjusted using a suitable organic or inorganic buffer,such as, for example, preferably using a phosphate buffer, tris buffer(tris(hydroxymethyl)aminomethane), HEPES buffer([4-(2-hydroxyethyl)piperazino]-ethanesulphonic acid) or MOPS buffer(3-morpholino-1-propanesulphonic acid). The choice of the respectivebuffer in general depends on the desired buffer molarity. Phosphatebuffer is suitable, for example, for injection and infusion solutions.

The medicament can be administered in a conventional manner, e.g. bymeans of oral dosage forms, such as, for example, tablets or capsules,by means of the mucous membranes, for example the nose or the oralcavity, in the form of dispositories implanted under the skin, by meansof injections, infusions or gels which contain the medicaments accordingto the invention. It is further possible to administer the medicamenttopically and locally in order to treat the particular joint disease asdescribed above, if appropriate, in the form of liposome complexes.Furthermore, the treatment can be carried out by means of a transdermaltherapeutic system (TTS), which makes possible a temporally controlledrelease of the medicaments. TTS are known for example, from EP 0 944 398A1, EP 0 916 336 A1, EP 0 889 723 A1 or EP 0 852 493 A1.

Injection solutions are in general used if only relatively small amountsof a solution or suspension, for example about 1 to about 20 ml, are tobe administered to the body. Infusion solutions are in general used if alarger amount of a solution or suspension, for example one or moreliters, are to be administered. Since, in contrast to the infusionsolution, only a few milliliters are administered in the case ofinjection solutions, small differences from the pH and from the osmoticpressure of the blood or the tissue fluid in the injection do not makethemselves noticeable or only make themselves noticeable to aninsignificant extent with respect to pain sensation. Dilution of theformulation according to the invention before use is therefore ingeneral not necessary. In the case of the administration of relativelylarge amounts, however, the formulation according to the inventionshould be diluted briefly before administration to such an extent thatan at least approximately isotonic solution is obtained. An example ofan isotonic solution is a 0.9% strength sodium chloride solution. In thecase of infusion, the dilution can be carried out, for example, usingsterile water while the administration can be carried out, for example,via a so-called bypass.

In another embodiment the TAFI-Ile347 or the TAFI-Ile347 gene can beused for the manufacturing of a diagnostic for the identification of avascular disease, in particular of a diabetic, including, withoutlimitation, an atrial fibrillation, stroke, prolonged intermittedneurological deficit (PRIND), transitory ischemic attack (TIA),atherosclerotic cerebrovascular disease (CVD) and/or coronary heartdisease.

For example, the TAFI-Ile347 can be used for the production of bindingproteins or binding peptides or aptamers, as described above, which canbe employed as diagnostic means. The TAFI-Ile347 gene can be useditself, in particular in its single stranded form, or for the productionof a complementary nucleic acid, preferably a complementarysingle-stranded DNA, which can be employed as a hybridization and/oramplification probe, generally known by a person skilled in the art.

Another embodiment of the present invention is directed to a method foradapting the dosage of a medicament for the treatment and/or prophylaxisof a thrombogenic disorder including, without limitation, includes anatrial fibrillation, stroke, prolonged intermitted neurological deficit(PRIND), transitory ischemic attack (TIA), atheroscleroticcerebrovascular disease (CVD) and/or coronary heart disease, wherein themethod comprises

-   (a) determining in a sample, e.g. from a diabetic, the presence of    an amino acid exchange from threonine to isoleucine at position 347    of the Thrombin-Activable Fibrinolysis Inhibitor (TAFI) with the    amino acid sequence according to SEQ ID NO: 1 (TAFI-Ile347), and-   (b) adapting the dosage of the medicament according to the result of    step (a).

As explained above, the TAFI-Ile347 shows an extended half-life and anenhanced antifibrinolytic activity in vitro and preferably an increasedrisk for stroke and TIA. Therefore, individuals with a TAFI-Ile347polymorphism need e.g. a higher amount of a medicament for the treatmentand/or prophylaxis of a thrombogenic disorder. In general, the dosage ofa medicament should advantageously be adapted to the genetic TAFIprofile of an individual or patient, in particular of individuals orpatients who are homozygous for isoleucine at position 347. Thedetermination step (a) of the present method can be carried out asexplained above.

The following Examples, Table and Figures shall explain the presentinvention without limiting the scope of the invention.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the protein sequence of TAFI.

FIG. 2 shows the nucleotide sequence of TAFI.

ABBREVIATIONS

TAFI variants having threonine at position 347 of the protein as aconsequence of polymorphisms at the corresponding position on bothalleles of the TAFI gene are called TAFI-347-TT.

TAFI variants having threonine and isoleucine at position 347 of theprotein as a consequence of a polymorphism at the corresponding positionon one of both alleles of the TAFI gene are called TAFI-347-TI.

TAFI variants having isoleucine at position 347 of the protein as aconsequence of polymorphisms at the corresponding position on bothalleles of the TAFI gene are called TAFI-347-II.

EXAMPLES

The TAFI polymorphism at the position 347 of the TAFI protein (NCBIaccession number for protein sequence: NP_(—)001863; FIG. 1) wasanalyzed in a patient cohort with or without cardiovascular events orendpoints.

1. SNP (Single Nucleotide Polymorphism) Detection by Sequencing andAnalysis

1.1 Amplification of Genomic DNA Region with Polymorphism of Interest

For the detection of the nucleotide exchange cytidine to thymidine atposition 1064 of the TAFI sequence with the NCBI accession numberNM_(—)001872 leading to TAFI-Ile347 protein variant (Table 2) thefollowing amplification primers were used:

(SEQ ID NO: 3) Forward primer: 5′-CACACCAGCTTTGCTACC-3′ (SEQ ID NO: 4)Reverse primer: 5′-CATTTTCCACTGTTTAGCTCC-3′

For the amplification the following PCR protocol was used, whereas allof the following reagents for the amplification were from AppliedBiosystems (Foster City, USA): 20 ng of genomic DNA; 1 unit of TaqGoldpolymerase; 1×Taq polymerase buffer; 500 μM of dNTP; 2.5 mM of MgCl₂;200 nM of each amplification primer pair (for sequence see Amplificationprimer pairs 1. above); H₂O ad 5 μl.

Amplification Program for PCR/Genotyping:

-   -   95° C.×10 min×1 cycle    -   95° C.×30 sec    -   70° C.×30 sec×2 cycles;    -   95° C.×30 sec    -   65° C.×30 sec×2 cycles;    -   95° C.×30 sec    -   60° C.×30 sec×2 cycles;    -   95° C.×30 sec    -   56° C.×30 sec    -   72° C.×30 sec×40 cycles;    -   72° C.×10 min    -   4° C.×30 sec×1 cycle;        1.2 Identification of Polymorphisms of Interest

For minisequencing and detection of polymorphisms the following protocolwas used, whereas all of the following reagents were from AppliedBiosystems (Foster City, USA). 2 μl of purified PCR product; 1.5 μlBigDye terminator kit; 200 nM of one sequencing primer (for sequence seeforward or reverse Amplification primer 1. above); H₂O ad 10 μl.

Amplification Program for Sequencing:

-   -   96° C.×2′×1 cycle;    -   96° C.×10″    -   55° C.×10″    -   65° C.×4′×30 cycles;    -   72° C.×7′    -   4° C.×30″×1 cycle;

The sequences were analyzed first with sequencing analysis tools(Applied Biosystems, Foster City, USA) for raw data extraction, then beprocessed with Phred (base caller), Phrap (assembler), polyphred (SNPcaller) and Consed (results viewer). Phred, Phrap, Polyphred and Consedare softwares designed at the University of Washington by Phil Green.

1.3 Statistical Approaches for Genotype/Phenotype Correlation

All analyses were performed with SAS statistical package (Version 6.12or higher, SAS Institute GmbH, Heidelberg/Germany). For the detection ofassociations between genetic polymorphisms and a large number ofclinical relevant parameters, descriptive statistics were computed(median, quartiles) and Wilcoxon-rank-sum-tests were performed.Wilcoxon-rank-sum-test was used for the comparison of two independentsamples. The computation of the test statistic is based on ranks in thepooled sample.

The search for associations between the SNPs and risk factors anddiseases, Chi-Square-Test were performed and numbers and percentageswere calculated to describe the data. The Chi-Square-Test is astatistical test for calculating the dependence of two variables. Thevalues of the variables are contained in two or more classes. To analyzethe association of those variables, a contingency table was used. Thistable contains as many rows as the number of realizations of the firstvariable and as many columns as the number of realizations of the secondvariable. Every cell contains a special patient's characteristic. Toconstruct a test statistic, the differences of calculated and observedfrequencies were computed.

After inspecting the results, relevant variables were selected. To takeaccount of confounding co-variables, logistic regression was used tovalidate the results. The logistic regression method was used to analyzethe influence of several explanatory variables on a certain responsevariable. The associated statistical test gave a p-value. Theinterpretation of this p-value is that there is a significant influenceof the associated explanatory variable.

For a binary variable, the odds ratio were calculated. The odds ratio isthe ratio of the odds that an event will occur in one group to the oddsthat the event will occur in the other group.

2. Results

The results were obtained by analyzing a subgroup of a patientpopulation whereas the subgroup had the following characteristics: alldiabetics, no treatment with the ACE inhibitor Ramipril, no alcohol.

TABLE TAFI 347 variants II TT/TI N % N % N total Stroke yes 5 7.35 193.35 24 no 63 92.25 549 96.65 612 TIA yes 3 4.41 4 0.7 7 no 65 95.59 56499.3 629

As can be seen from the Table, all individuals with TAFI-I347Ipolymorphism have an increased risk for stroke (7.35% vs. 3.35%) and TIA(4.41% vs. 0.7%) compared to individuals with TAFI-I347T and TAFI-T347Tpolymorphism.

The invention claimed is:
 1. A method for adapting the dosage of amedicament for the treatment of a thrombogenic disorder in a patient,wherein the method comprises: (a) determining in a sample from thepatient the presence of an amino acid exchange from threonine toisoleucine at position 347 of the Thrombin-Activable FibrinolysisInhibitor (TAFI) with the amino acid sequence according to SEQ ID NO: 1(TAFI-Ile347) by analyzing the amino acid sequence at position 347 ofTAFI protein in said sample or by analyzing the nucleic acid sequence ofTAFI gene at position 1064 of SEQ ID NO: 2 in said sample, and (b)increasing the dosage of the medicament if said amino acid exchange ofstep (a) is present.
 2. The method according to claim 1, wherein thethrombogenic disorder is selected from the group consisting of atrialfibrillation, stroke, prolonged intermitted neurological deficit(PRIND), transitory ischemic attack (TIA), atheroscleroticcerebrovascular disease (CVD) and coronary heart disease.
 3. The methodaccording to claim 1 wherein the sample is from a diabetic patient.